Water contributes to many aspects of protein function; however, difficulties associated with experimentally measuring protein-water interactions with site-resolution has left our understanding of the nature of protein hydration rather unclear. Though solution nuclear magnetic resonance (NMR) spectroscopy has been proposed as a means to characterize these interactions, several features of the interaction of water with the protein render the approach insensitive and susceptible to artifacts. We have developed reverse micelle encapsulation as a means to overcome these limitations, thereby permitting the unambiguous differentiation of ratios of the nuclear Overhauser effect (NOE) and rotating frame Overhauser effect (ROE) to characterize hydration water dynamics. Recently, we have shown that this approach can be used to survey the surface hydration dynamics of proteins using ubiquitin encapsulated in sodium bis(2-ethylhexyl) sulfosuccinate (AOT) reverse micelles. We find that surface hydration dynamics are heterogeneous and clustered suggesting that local factors contribute to differential hydration dynamics on the protein surface. Furthermore, there is a correlation between surfaces having slow hydration water and whether that surface participates in protein- protein interactions involving a dry interface suggesting tha the hydration shell may contribute to molecular recognition events. In this proposal we extend our analysis to measure the hydration dynamics of representative members of the PSD-95/disc-large/zonula occludens-1 (PDZ) domain family. PDZ domains are recognition modules in protein signaling pathways. These small, structurally homologous proteins have been extensively characterized biochemically and biophysically. These factors make PDZ domains an ideal system for measuring hydration dynamics using high-resolution NMR spectroscopy. The goals of this proposal are two-fold: 1) To understand what aspects of the protein surface correspond to differential hydration dynamics and 2) to investigate the role of hydration dynamics on ligand binding. We will use high resolution NOESY- HSQC and ROESY-HSQCs to extract site specific NOE/ROE ratios that report on surface hydration. We will measure surface hydration dynamics of several unliganded PDZ domains to understand how local amino acid chemistry may contribute to strength of water-protein interactions. Additionally, we will analyze the surface hydration dynamics of unliganded and liganded states to determine whether hydration dynamics are conserved between structurally related proteins. Together we seek to enhance our understanding of the molecular underpinnings of the protein hydration shell and how it may contribute to molecular recognition.